**4. Plasma membrane events following KATP closure**

Surprisingly, the plasma membrane of β-cells contains up to 60 channels of 16 ion channel families [69]. Moreover, ion channels are also located on the membrane of IGVs to facilitate fusion with the plasma membrane and insulin exocytosis. Resting plasma membrane potential (*V*p) is created predominantly by the activity of K+ -channels due to a higher concentration of K+ inside the β-cell (~150 mM), exceeding that one established outside in capillaries or interstitial fluid (~5 mM). Experimentally, *V*p values are measured to be approximately of – 75 mV [70]. The KATP closure then induces depolarization [69, 71–73] and activation of CaL [74]. The action potential firing is the entity that activates CaL-KV cycles (in rodents), however this firing is initiated by more channel types.

Surprisingly, the action potential firing is not induced until >90% of KATP channels are closed [75, 76]. As a result, only the closure of the remaining ~10% of the KATP population leads to depolarization [76]. In fact, the activity of the whole KATP population decreases exponentially with the increasing glucose concentration. Interestingly, 50% of the KATP population is already closed at 2–3 mM glucose, while *V*p remains steady. However, at about 7 mM glucose, 100% of the KATP ensemble is closed. This is being reflected by the completely vanished KATP current, which leads to action potential firing [69, 70]. This event is termed as a supra-threshold depolarization.

Thus, hyperpolarized interburst phases are induced, while a nearly permanent firing exists at high >25 mM glucose [70]. An intermediate depolarization at 10 mM glucose was reported for mouse β-cells, reversed upon withdrawal of Ca2+ and Na+ , supporting the participation of other channels, such as nonspecific cation channels, contributing to the depolarization (inward) flux [45]. Even an efflux of Cl<sup>−</sup> was suggested to fulfil this role [77], including the opening of LRRC8/VRAC anion channels [78, 79]. The participation of TRPM4 and TRPM5 [80] providing inward currents of certain levels seem to be required for induction of sufficient membrane depolarization together with KATP closing [46]. This is because the measured resting *V*p of – 75 to −70 mV is already depolarized by a some extent from the equilibrium *V*pequi of −82 mV (5 mM vs. 130 mM [K+ ]). The shift is probably due to the opening of nonspecific cation channels, since any of Na<sup>+</sup> , Ca2+ and K+ can penetrate them. The 100% KATP closing at higher glucose causes only an insufficient depolarization. Without nonspecific cation channels (or Cl<sup>−</sup> channels), the established *V*p would only be equal to *V*pequi, so any shift to −50 mV required for CaL would not take place. Contribution by the basal opening of other synergic channels is therefore essential. Open synergic channels always induce the inward shift in *V*p, so to that depolarization given by 100% KATP closing reaches −50 mV. This allows opening of CaL and action potential firing. In summary, besides the heat-activated TRPV1 channel (capsaicin receptor), and TRPV2 or TRPV4, the H2O2-activated TRPM2 [2], or Ca2+-activated TRPM4 and TRPM5 channels belong to the important group of possible synergic channels expressed in β-cells [46].

The same reasoning concerns with anion channels, particularly Cl<sup>−</sup> channels. The active Cl− transport is provided in β-cells by SLC12A, SLC4A, and SlC26A channels. These channels set the cytosolic Cl− concentration above thermodynamic equilibrium. Besides GABAA, GABAB and glycine receptor Cl<sup>−</sup> channels considered to be part of the insulin secretion machinery, also volume-regulated anion channels (VRAC) were shown to be open at high glucose. VRACs are heteromers of the leucine-rich repeat containing 8 isoform A (LRRC8A) with other LLRC8 isoforms, forming anion channels [79]. Ablation of LRRC8 in mice led to delayed Ca2+ responses of β-cells to glucose and diminished GSIS in mice, demonstrating the modulatory role of LRRC8A/VRAC on membrane depolarization leading to CaL responses [78, 79].

**39**

to ATP inhibition [91].

*Redox Signaling is Essential for Insulin Secretion DOI: http://dx.doi.org/10.5772/intechopen.94312*

delayed rectifier K+

Upon the action potential firing thus metabolically driven *V*p oscillations occur due to the initial glucose rising [69, 70]. Cytosolic Ca2+ oscillations are superimposed from fast (2–60 s periods) and slow (up to several min) Ca2+ oscillations [81], stemming from *V*p oscillations and an interplay with Ca2+ efflux from the endoplasmic reticulum (ER) [82]. Collectively they lead to pulsatile insulin secretion. The ER involvement is given by the phospholipase C (PLC), responding to the glucosestimulated Ca2+ influx. PLC produces inositol triphosphate (IP3), which opens the Ca2+ channel of IP3 receptor (IP3R) of ER; plus diacylglycerol (DAG). Importantly, DAG permits the opening of TRPM4 and TRPM5 via the protein kinase C (PKC) pathway. Another ER Ca2+ channel, the ryanodine receptor (RyR) may also participate, being activated by ATP, fructose, long-chain acyl-CoAs and cyclic adenosine 5′-diphosphate ribose [81]. Also, the role of other channels was demonstrated for permitting store-operated Ca2+ entry from ER, particularly of the ternary complex of TRPC1/Orai1/STIM1 [46, 83]. TRPC1 belongs to the transient receptor potential canonical (TRPC) family with a modest Ca2+ selectivity. TRPC1 interacts with Orai1 [84], and in such a functional complex, its channels are activated by STIM1,

affecting the amplitude of Ca2+ oscillations, and correlating with GSIS.

dependent channels (KV) in rodents [52] or calcium-dependent (KCa) K+

**5. Possible redox regulations of KATP and other channels**

As mentioned above, deactivation of CaL is ensured by the opening of voltage-

humans. Among the former, tetrameric KV2.1 is the prevalent form in rodent β-cells. A

ing of KV2.1 channels repolarizes *V*p and thus closes CaL channels. Ablation of KV2.1 thus reduces Kv currents by ~80% and prolongs the duration of the action potential, so more insulin is secreted. Mice with ablated KV2.1 possess lower fasting glycemia but elevated insulin and reportedly improved GSIS [86]. In contrast, human β-cells use KCa1.1 channels (i.e. BK channels) for repolarization of *V*p [70]. Note also that downregulation of KV was observed after islet incubation with high glucose for 24 hr [87].

The structure of KATP has been resolved and numerous mutagenesis studies of KATP have been conducted. Amino acid residues that are candidate redox targets are yet to be identified. The KATP channel is a hetero-octamer consisting of four external regulatory sulfonylurea receptor 1 (SUR1, a product of *Abcc8* gene) subunits and four pore-forming subunits of potassium inward rectifier, Kir6.2 (*Kcnj11* gene) [88, 89]. These Kir6.2 subunits cluster in the middle of ~18 nm size structure with a ~13 nm height [90]. The part exposed to the cytosol contains an ATP binding site, located about 2 nm below the membrane. A single ATP molecule was reported to close the channel, i.e. with the other three binding sites left unoccupied [91]. However, the ATP binding site overlaps with the binding site for phosphatidylinositol 4,5-bisphosphate (PIP2), which stabilizes the open state. Palmitoylation of Cys166 of Kir6.2 was then reported to amplify the responsiveness to PIP2 [92]. Upon the release of PIP2 from the binding site, the open probability becomes decreased [90, 93, 94].

Diazoxide or cromakalim, as well as numerous other openers, set KATP pharmacologically in the open state even at a high ATP concentration [95]. In contrast, the artificial KATP closing by sulfonylurea derivatives, such as glibenclamide, takes place independently of ATP. Besides this sulfonylurea binding site, each of the four SUR1 subunits contains MgATP and MgADP binding sites. MgATP is hydrolyzed at the nucleotide binding fold 1 (NBF1) to MgADP. Resulting MgADP subsequently activates KATP at NBF2. This is indeed reflected by the ATP-sensitive increase in K<sup>+</sup> conductance and following lower excitability, accompanied by the lower sensitivity



#### *Redox Signaling is Essential for Insulin Secretion DOI: http://dx.doi.org/10.5772/intechopen.94312*

*Type 2 Diabetes - From Pathophysiology to Cyber Systems*

of K+

**4. Plasma membrane events following KATP closure**


however this firing is initiated by more channel types.

*V*pequi of −82 mV (5 mM vs. 130 mM [K+

of nonspecific cation channels, since any of Na<sup>+</sup>

possible synergic channels expressed in β-cells [46].

equilibrium. Besides GABAA, GABAB and glycine receptor Cl<sup>−</sup>

channels. These channels set the cytosolic Cl−

Without nonspecific cation channels (or Cl<sup>−</sup>

Surprisingly, the plasma membrane of β-cells contains up to 60 channels of 16 ion channel families [69]. Moreover, ion channels are also located on the membrane of IGVs to facilitate fusion with the plasma membrane and insulin exocytosis. Resting plasma membrane potential (*V*p) is created predominantly by the activity

exceeding that one established outside in capillaries or interstitial fluid (~5 mM). Experimentally, *V*p values are measured to be approximately of – 75 mV [70]. The KATP closure then induces depolarization [69, 71–73] and activation of CaL [74]. The action potential firing is the entity that activates CaL-KV cycles (in rodents),

are closed [75, 76]. As a result, only the closure of the remaining ~10% of the KATP population leads to depolarization [76]. In fact, the activity of the whole KATP population decreases exponentially with the increasing glucose concentration. Interestingly, 50% of the KATP population is already closed at 2–3 mM glucose, while *V*p remains steady. However, at about 7 mM glucose, 100% of the KATP ensemble is closed. This is being reflected by the completely vanished KATP current, which leads to action potential firing [69, 70]. This event is termed as a supra-threshold depolarization. Thus, hyperpolarized interburst phases are induced, while a nearly permanent firing exists at high >25 mM glucose [70]. An intermediate depolarization at 10 mM glucose was reported for mouse β-cells, reversed upon withdrawal of Ca2+ and Na+

supporting the participation of other channels, such as nonspecific cation channels,

The 100% KATP closing at higher glucose causes only an insufficient depolarization.

only be equal to *V*pequi, so any shift to −50 mV required for CaL would not take place. Contribution by the basal opening of other synergic channels is therefore essential. Open synergic channels always induce the inward shift in *V*p, so to that depolarization given by 100% KATP closing reaches −50 mV. This allows opening of CaL and action potential firing. In summary, besides the heat-activated TRPV1 channel (capsaicin receptor), and TRPV2 or TRPV4, the H2O2-activated TRPM2 [2], or Ca2+-activated TRPM4 and TRPM5 channels belong to the important group of

The same reasoning concerns with anion channels, particularly Cl<sup>−</sup>

ered to be part of the insulin secretion machinery, also volume-regulated anion channels (VRAC) were shown to be open at high glucose. VRACs are heteromers of the leucine-rich repeat containing 8 isoform A (LRRC8A) with other LLRC8 isoforms, forming anion channels [79]. Ablation of LRRC8 in mice led to delayed Ca2+ responses of β-cells to glucose and diminished GSIS in mice, demonstrating the modulatory role of LRRC8A/VRAC on membrane depolarization leading to CaL

transport is provided in β-cells by SLC12A, SLC4A, and SlC26A

contributing to the depolarization (inward) flux [45]. Even an efflux of Cl<sup>−</sup>

suggested to fulfil this role [77], including the opening of LRRC8/VRAC anion channels [78, 79]. The participation of TRPM4 and TRPM5 [80] providing inward currents of certain levels seem to be required for induction of sufficient membrane depolarization together with KATP closing [46]. This is because the measured resting *V*p of – 75 to −70 mV is already depolarized by a some extent from the equilibrium

Surprisingly, the action potential firing is not induced until >90% of KATP channels

inside the β-cell (~150 mM),

]). The shift is probably due to the opening

channels), the established *V*p would

concentration above thermodynamic

channels consid-

can penetrate them.

channels.

, Ca2+ and K+

,

was

**38**

The active Cl−

responses [78, 79].

Upon the action potential firing thus metabolically driven *V*p oscillations occur due to the initial glucose rising [69, 70]. Cytosolic Ca2+ oscillations are superimposed from fast (2–60 s periods) and slow (up to several min) Ca2+ oscillations [81], stemming from *V*p oscillations and an interplay with Ca2+ efflux from the endoplasmic reticulum (ER) [82]. Collectively they lead to pulsatile insulin secretion. The ER involvement is given by the phospholipase C (PLC), responding to the glucosestimulated Ca2+ influx. PLC produces inositol triphosphate (IP3), which opens the Ca2+ channel of IP3 receptor (IP3R) of ER; plus diacylglycerol (DAG). Importantly, DAG permits the opening of TRPM4 and TRPM5 via the protein kinase C (PKC) pathway. Another ER Ca2+ channel, the ryanodine receptor (RyR) may also participate, being activated by ATP, fructose, long-chain acyl-CoAs and cyclic adenosine 5′-diphosphate ribose [81]. Also, the role of other channels was demonstrated for permitting store-operated Ca2+ entry from ER, particularly of the ternary complex of TRPC1/Orai1/STIM1 [46, 83]. TRPC1 belongs to the transient receptor potential canonical (TRPC) family with a modest Ca2+ selectivity. TRPC1 interacts with Orai1 [84], and in such a functional complex, its channels are activated by STIM1, affecting the amplitude of Ca2+ oscillations, and correlating with GSIS.

As mentioned above, deactivation of CaL is ensured by the opening of voltagedependent channels (KV) in rodents [52] or calcium-dependent (KCa) K+ -channels in humans. Among the former, tetrameric KV2.1 is the prevalent form in rodent β-cells. A delayed rectifier K+ -current is induced at positive *V*p down to −30 mV [85]. The opening of KV2.1 channels repolarizes *V*p and thus closes CaL channels. Ablation of KV2.1 thus reduces Kv currents by ~80% and prolongs the duration of the action potential, so more insulin is secreted. Mice with ablated KV2.1 possess lower fasting glycemia but elevated insulin and reportedly improved GSIS [86]. In contrast, human β-cells use KCa1.1 channels (i.e. BK channels) for repolarization of *V*p [70]. Note also that downregulation of KV was observed after islet incubation with high glucose for 24 hr [87].
